The present invention relates to a method for the preparation of a physiologically active, water-soluble hemin arginate or hemin lysinate complex intended for use in tablets or capsules or as a dry substance for injection after reconstitution, e.g., with sterile saline solution.
Hemin occurs in organisms as a prosthetic group of hemoglobin in most cytochromes and in certain enzymes. Hemoglobin is synthesized in the bone marrow. When hemin proteins decompose, hemin is released, but only a minor part of it is used in the synthesis of new hemin proteins under normal physiological conditions. Hemin is split by the action of hemin oxygenase into biliverdin, which is further reduced to bilirubin. Native, intact hemoglobin does not, naturally, serve as a substrate for hemin oxygenase.
Defects in the hemoglobin synthesis may be due to disturbed synthesis of hemin or of globin chains. The hemin synthesis may be disturbed because of: (a) lack of some component necessary for the synthesis, or (b) dysfunction of an enzyme catalyzing the synthesis.
Iron deficiency is the limiting factor in the hemin synthesis. The organism gets its daily requirement of iron (1-2 mg) with food. Iron deficiency may be due to a diet deficient in iron or possibly to the presence of iron-building compounds in the food. Disturbances in the iron absorption mechanism may also lead to iron deficiency, despite adequate iron content in the food. Regardless of the cause, iron deficiency sooner or later leads to anemia.
In the rarely occurring vitamin B.sub.6 deficiency, the absorption of iron is normal, but its utilization by the cells is inhibited. As a consequence of this, a certain type of sideroblastic anemia develops. Iron deficiency anemia is treated either with oral iron preparations, e.g., iron sulphate or iron gluconate, or, more rarely, with injections such as iron sorbitol. When the iron absorption mechanism is disturbed, these conventional oral preparations are useless; the iron does not even penetrate into the cells of the intestinal mucosa. In contrast to inorganic iron, hemin iron, in which the iron is bound to hemin, is absorbed by these cells even in such cases of disturbed iron absorption which are resistant to conventional oral therapy. Thus, hemin iron is the only known, effective remedy for oral treatment of therapy-resistant cases. Hemin iron has been found to be four to five times better absorbed than inorganic iron, even in quite healthy subjects (Seppanen H & Takkunen H: Suomen Laakarilehti 36:2071-2072, 1981).
The synthesis of hemin is enzymatically regulated. Impaired function of the enzymes catalyzing the hemin synthesis may be either hereditary or due to external factors. It invariably leads to decreased formation of hemin, manifested by the development of porphyria or certain kinds of sideroblastic anemia or other diseases.
Porphyria is the most important group of diseases resulting from impaired enzyme function. In porphyria patients there is an accumulation of porphyrins, intermediary products in the hemin synthesis, and an increased excretion of these into urine and feces. Most kinds of porphyria are manifested by acute attacks which are extremely difficult to master.
Sometimes, sideroblastic anemias of different kinds may develop instead of porphyria as a consequence of dysfunction of enzymes participating in the hemin synthesis. Sideroblastic anemias, too, may be either hereditary or acquired.
The treatment of porphyria has until now been based principally on the avoidance of certain drugs and the administration of large amounts of carbon hydrates during the acute attacks, but the effect has been poor. Since the etiology of porphyria became clarified, intravenous treatment with hemin compounds (hematin) has been continuously gaining ground. Hematin has proved effective in the treatment of porphyria attacks, but in more than 50% of the patients it has caused thrombophlebitis. Moreover, it is very unstable and therefore unsuitable for production on an industrial scale. There are, thus, very few possibilities for effective treatment of porphyria patients.
The aim of the present invention is to produce a water-soluble hemin iron compound for treatment of certain kinds of anemia, with the iron ready at hand, so to speak, in the hemin molecule. The compound is intended, in the first place, for treatment of porphyria, where the normal production of hemoglobin is disturbed for some reason or other. As the compound is intended for oral administration in tablets or capsules as well as for injection, it must be water-soluble.
Hemin, which is sparingly soluble in water, can be obtained in pure form from blood by extraction with a mixture of hydrochloric or acetic acid from a water solution of hemolyzing erythrocytes. Another method is based on the extraction of hemin with acetone in the presence of, e.g., histidylhistidine, pilocarpine, or imidazole at pH 7.0 (Wakid N. W. & Helou K. Y. : Int. J. Biochem. 4:259-267, 1973).
PCT patent application Ser. No. 813749 (PCT/FI81/00026) describes a method for the preparation of a water-soluble hemin concentrate in which about 40% w/w is hemin and the rest is a "blood substance" of unknown nature. The product is intended for use in lyophilized form as an iron supplement in food or as an antianemic drug.
The drawback of this method is that the final product is a mixture of hemin and "blood substance." As the latter component is not uniform, the mixture is unsuitable for injection.
The above-referenced PCT patent application corresponds to U.S. Pat. No. 4,431,581, Lindroos, issued Feb. 15, 1984. Not only are the products of this Lindroos patent not intended for injection purposes, as are the water-soluble materials of the present invention, but the materials of the Lindroos patent could not be used for injection purposes because of the high risk of life threatening anaphylactic shock, if an attempt were made to use them as injectable materials. Nothing set forth in the Lindroos patent would indicate that they are useful for such purposes, and, in fact, a review of the process conditions employed in Lindroos and analytical testing of the product indicates that the products of Lindroos are totally different from the products of the present invention. For example, Lindroos operates at a high pH, where heme easily degrades; in accordance with the present invention, reaction is carried out in the presence of amino acids which are weakly basic.
A sample of the Lindroos product, in accordance with U. S. Pat. No. 4,431,581 was acquired from a licensee of a corresponding patent. The iron content of the heme concentrate of Lindroos was found to be 2.94%, while a product made in accordance with the present invention had an iron content of 4.74%. Still further, the iron of the Lindroos patent in the heme concentrate is in the +2 oxidation state, while the iron in the material of the present invention is in the +3 oxidation state. A measurement of the pH of a 1% water solution of the Lindroos patent was found to be 8.78, while a similar test carried out on the product of the present invention, hemin arginate, in a 1% water solution was found to be 8.55. Of further significance, spectra were run of both the Lindroos product and the hemin arginate product of the present invention by infrared, ultraviolet, and hydrogen nmr. A comparison of the nmr spectra of the two products clearly shows two totally different products, a comparison confirmed by a study of the ultraviolet and infrared spectra.
A comparison was also made between the product of the present invention and that described in Livshits et al, Zh.Org.Khim 1977, 13(2), page 436 (Chem. Abstracts Vol. 87:53549 p. 1977). A comparison of the spectra shown in FIG. 2b on page 440 of the referenced Livshits article with the spectra described above for the material of the present invention showed two completely different products. Further, Livshits et al describes the synthesis of amides where the carboxylic acid groups of heme, via normal methods for peptide synthesis, have been coupled to lysine and some derivatives of this amino acid so that a strong covalent bond is formed. To precipitate heme from this compound, hydrolysis is required, which can be accomplished either enzymatically or by boiling for several hours in strong acid solution. By contrast, the present invention describes a complex salt of heme and amino acid where the binding is electrovalent, so that it has been shown that heme may be precipitated merely by acidifying a solution of the product.
Porphyria has been treated in hospitals with a mixture prepared extempore by dissolving hemin in a sterile sodium carbonate solution (hematin). As this solution is unstable it cannot be manufactured as a commercial product on a large scale. Moreover, hematin causes thrombophlebitis at the site of injection in about 50% of the cases, probably because of the high pH of the solution. This is a serious drawback which reduces the usefulness of the product considerably.